Pseudomonas fluorescens F113 and Pseudomonas putida KT2440. This protein was previously identified as a master regulator of flagella and biofilm formation in both strains. This work has demonstrated that FleQ from both bacteria are conserved and functionally equivalent for motility regulation. Furthermore, the ChIP-seq analysis has shown that FleQ is a global regulator with the identification of 121 and 103 FleQ putative binding sites in P. fluorescens F113 and P. putida KT2440 respectively. Putative genes regulated by FleQ included, as expected, flagellar and motility-related genes and others involved in adhesion and exopolysaccharide production. Surprisingly, the ChIP-seq analysis also identified iron homeostasis-related genes for which positive regulation was shown by RT-qPCR. The results also showed that FleQ from P. fluorescens F113 shares an important part of its direct regulon with AmrZ, a global regulator also implicated in environmental adaption. Although AmrZ also regulates motility and iron uptake, the overlap occurred mostly with the iron-related genes, since both regulators control a different set of motility-related genes.Bacterial motility plays a crucial role in competitiveness and colonization in the rhizosphere. In this work, Chromatin ImmunoPrecipitation Sequencing (ChIP-seq) analysis has been used to identify genes putatively regulated by the transcriptional regulatory protein FleQ in  In this regulatory cascade, the transcriptional regulator FleQ appears to be the master regulator2. The function of FleQ in flagella synthesis regulation has been studied in Pseudomonas aeruginosa2, in P. putida5 and in P. fluorescens8. In these species, mutations in the fleQ gene result in non-motile, aflagellated bacteria. FleQ is an atypical enhancer binding protein (EBP) from the NtrC family of bacterial transcription factors (TFs) with three fundamental domains: a N-terminal REC domain which lacks the aspartic acid that serves as a phosphorylation site in other members of the same family, a central AAA+/ATPase σ54 (RpoN)-interaction domain and a C-terminal helix-turn-helix DNA-binding domain9. It has been described that FleQ is able to activate the expression of genes involved in flagellar export (flhA and fliLMNOPQ operon), localization and regulation of the flagellar apparatus (flhF and fleN), structural components of the flagellar basal body and motor switch complex (fliEFG) and the fleSR genes10. In the regulation of flagellar operons, FleQ works along with the alternative σ factor RpoN2. It also works together with the anti-activator FleN, another ATPase, by means of direct protein-protein interactions15. It has also been shown that FleQ can specifically bind the bacterial second messenger cyclic di-guanosine monophosphate (c-di-GMP)17 and the crystal structure of FleQ bound to c-di-GMP has been resolved18. Interestingly, most of the flagellar genes are moderately regulated by c-di-GMP, showing a downregulation when the intracellular level of this molecule is high13.Flagella biosynthesis in pseudomonads requires more than 50 genes subjected to four levels of hierarchical regulationP. aeruginosa exopolysaccharides  in a c-di-GMP-dependent manner, triggering either the activation or repression of these genes18. In the regulation of these operons, FleQ does not rely on RpoN but on the vegetative sigma factor (σ70)14. In the case of the pel operon the mechanism proposed incorporating structural and functional data implies that FleQ binds to two sites in the promoter of the operon but the effect on gene expression depends on the level of c-di-GMP. Without c-di-GMP, a hexamer of FleQ, although bound to two sites, relies on one of the sites (FleQ box 2) to repress gene expression when bound to FleN in presence of ATP. On the other hand, in response to c-di-GMP, the FleN/FleQ/DNA complex suffers a conformational change turning the FleQ multimer into an activator from the other promoter site (FleQ box 1)14. In addition, FleQ has been recently described as regulator of two strain-specific EPSs in P. putida KT2440, Pea and Peb17, the first being a key element of biofilm formation in this bacterium21. Other polysaccharide regulated by FleQ in a c-di-GMP dependent mode in P. putida is cellulose, through transcriptional regulation of the bcs operon22. FleQ has also been shown to regulate the expression of the cdrA and lapA genes, encoding adhesins required for biofilm formation in P. aeruginosa23 and P. putida24 respectively, in a c-di-GMP-dependent way. Furthermore, FleQ has been shown to be essential for biofilm formation in P. putida26.Besides the flagellar operons, FleQ regulates the biosynthesis of 28. A ChIP-seq assay in P. fluorescens F113 showed that at least 215 genes were putatively regulated by AmrZ. AmrZ was shown to regulate genes required for iron homeostasis, synthesis and degradation of c-di-GMP and motility28. Similar results were obtained in P. aeruginosa29. AmrZ is an important determinant of c-di-GMP levels. In F113, AmrZ transcriptionally regulates multiple genes encoding diguanylate cyclases and the amrZ mutant shows enhanced motility, altered exopolysaccharides production, reduced biofilm formation, lack of rhizosphere colonization competence and reduced cytoplasmic levels of c-di-GMP30. It is important to note that AmrZ strongly represses the expression of the fleQ gene in both, P. aeruginosa and P. fluorescens species31.Another central node in environmental adaption in pseudomonads is the transcriptional regulator AmrZP. fluorescens F113 and P. putida KT2440 by using ChIP-seq and to analyze the possible overlap between the FleQ and AmrZ regulons in P. fluorescens F113.Considering that FleQ and AmrZ regulate similar traits such as motility, exopolysaccharides production and biofilm formation, the aim of this work was to identify the genes and operons regulated by FleQ in fleQ mutants are non-motile because they lack flagella. In order to determine the functionality of HA-FleQ fusion proteins to be used for ChIP-seq, we complemented the swimming motility phenotype of fleQ mutant derivatives of F113 and KT2400 with their respective cloned fusion genes. As shown in Fig. fleQ mutants in both strains. Figure KT2440 was also able to complement the motility of the F113 fleQ mutant and HA-FleQF113 complemented the KT2440 fleQ mutant. These results show not only the functionality of the HA fusions, but also that FleQ proteins from both species are functionally equivalent, at least in the regulation of motility.FleQ is the master regulator for flagella synthesis in pseudomonads and F113 in Pseudomonas fluorescens F113 and HA-FleQKT2440 in P. putida KT2440) were performed, yielding 10 ng of immunoprecipitated DNA. Immunoprecipitated DNA from the four replicas was pooled and subjected to Illumina sequencing. After quality filtering, 6,653,248 reads  in the case of F113 and 20,558,997  in the case of KT2440 of 50 nts length were used for subsequent experiments. In the case of F113, bioinformatic analysis yielded 496 peaks distributed along all the genome  and as a repressor for the expression of genes implicated in exopolysaccharides production  and others, both in P. fluorescens F113 and in P. putida KT2440. Interestingly, amrZ was negatively regulated by FleQ both in P. fluorescens F113 and in P. putida KT2440.Gene expression analysis was performed in both species for a selected group of genes that have a peak in their promoter region. As shown in Fig. bifA- background was used for high c-di-GMP levels and a sadC-wspR- for low levels of the second messenger32. A bifA- background was used in KT2440 for elevated c-di-GMP in comparison to the WT33. The results were not significantly different from those in the wild-type strains. In the case of F113, 126 out of the 159 genes identified as putatively regulated in the wild-type strain, were also identified in the bifA- assay and 106 in the sadC-wspR- assay. One hundred and eighty are common between bifA- and sadC-wspR-, that have extreme levels of c-di-GMP. In the case of P. putida KT2440, in the bifA- background, 149 peaks with a fold enrichment higher than 5 were detected. 107 genes were coincident with the genes in the wild-type assay. These genes include all of the genes implicated in c-di-GMP turnover and exopolysaccharide production and most of the genes implicated in motility and iron homeostasis. It also included genes such as lapA and amrZ. Furthermore, ten other genes that appeared as potential FleQ targets in the wild-type assay, were also identified in the bifA- background, although the peaks ranged between four and five-fold. These results clearly show that c-di-GMP does not play a relevant role in the binding of FleQ to promoters which is in agreement to the current proposed model of regulation18. Supplementary Tables Since regulation by FleQ may be influenced by the second messenger c-di-GMP, similar ChIP-seq experiments to those reported above were performed in F113 and KT2440 backgrounds with altered c-di-GMP levels. For F113, a flgF, flgG, PSF113_1540, fliC, flaG, fliD, fliST, fleQ, fleSR, fliE, fliK, fliLM, flhA, flhF , “c-di-GMP”  and “virulence” . The remaining genes were included in “others” category  or their functions are unknown . In order to test the relevance of FleQ as a regulator of iron homeostasis, we analyzed the expression of several iron related genes in iron deficient conditions, in both backgrounds, in the wild-type strain and in the −034 in F113. To avoid iron bias those regions containing a Fur motif were removed from the dataset, resulting in 62 peaks that were, again, analyzed with the same tools. Once more for this particular situation, it was not possible to obtain a FleQ DNA-binding consensus motif, as sequences corresponding to the IHF and σ54 binding sites masked any other possible conserved sequence. Similar results were found with the KT2440 peaks. In view of the fact that no conserved region for FleQ binding site could be found, a FleQ consensus sequence (GTCaNTAAAtTGAC) that has been proposed for P. aeruginosa23, was searched with MAST, BLASTn and FIMO in P. fluorescens F113. The genes that included this P. aeruginosa motif, such as lapA-like, PSF113_1970, fliL, fliE and flhA, were found in our F113 analysis and corresponded to peaks. However, a total of 536 matches were detected in the F113 genome, 490 of them with a p-value below the 1e-5 range . Most of the matches corresponded to regions that were not present in our ChIP-seq output. Consequently, we were not able to define a robust FleQ consensus binding sequence in P. fluorescens F113. More recently, a consensus sequence for the FleQ binding site in P. putida KT2440 (GTCAaAAAAtTGAC) was proposed17 based on the promoter regions of 15 selected genes and the previously proposed consensus for P. aeruginosa. The genes included fliE, lapA, algD, bcs, pea, peb, fleS, flhA. Similarly to F113, the FleQ binding site in KT2440 was searched in the pool of 103 peaks obtained in the KT2440 ChIP-seq assay using FIMO and MAST tools. As a result, 18 matches were found  and were attributed to 15 peaks, as in some peaks the motif appeared more than once. The 15 peaks were assigned to 15 genes. Five genes (29.5%) were classified in the “cell wall” category while the remaining 10 genes were distributed in “motility/chemotaxis” (fliE and flhB), “iron”, “transport”, “regulation/signal transduction” (amrZ), “others” and “unknown” categories. Genes included in “cell wall” functional class were lapA, algD and genes of the pea, peb and bcs operons, all of them previously identified in vitro17. The same coincidence was observed with fliE. The motif was found three times in the case of lapA and twice in pea. Although this motif seems to be congruent in a specific set of genes, being most of them related with exopolysaccharide synthesis, we were unable to propose a consensus sequence that might expand to a majority of the genes identified as being regulated by FleQ in P. putida KT2440. Evaluation of intergenic peaks independently did not provide further information in the search for the union consensus sequence in any of the strains.With the purpose of finding a specific motif binding site for FleQ, the summit positions of the 121 peaks in the F113 ChIP-seq assay and 103 peaks for KT2440 and a region of 100 nts on each side were introduced in the MEME tool. However, it was not possible to determine a robust motif for FleQ as many peaks were located in the promoter region of iron-responding genes in both cases. Therefore, the main resulting motif was the iron responsive Fur-box motif (not shown). This motif was present in 44 of the peaks with an e-value of 1.5eamrZ gene appears to be repressed by FleQ both in P. fluorescens and in P. putida. Since AmrZ has been shown to be a global motility and iron regulator in F113, the 159 genes putatively regulated by FleQ in this strain were compared with the 215 genes found to be putatively regulated by AmrZ28. The results showed an overlap of 45 genes putatively regulated by both proteins in P. fluorescens F113 , cellulose-like (bcs) and two less characterized polysaccharides, Pea and Peb21. It is known that FleQ binds in vitro to the promoter region of the gene clusters encoding the synthesis of Bcs, Pea and Peb and that this regulator strongly represses the synthesis of alginate under cell wall stress conditions17. Our in vivo experiments have validated the binding of FleQ to the bcs, alg and peb promoters, since peaks have been detected in these locations. We have not detected a peak upstream of PP_3132, the first gene in the pea operon. However, a strong peak was found close to this location, upstream of PP_3126, encoding a “polysaccharide biosynthesis/export protein” that might have a double functionality in the production of the Pea polysaccharide. We have also confirmed in this work that FleQ is a strong repressor of bcsA expression. Regarding biofilm formation, FleQ has been shown to positively regulate the expression of the lapA gene, which encodes a large adhesin essential for biofilm formation. Positive regulation of lapA and lapA-like genes has been shown in P. putida24 and P. aeruginosa23. We have confirmed here the in vivo binding of FleQ to the lapA promoter and the transcriptional activation of this gene by the regulator in P. putida. We have extended this observation to P. fluorescens by showing that in F113, FleQ also binds to the lapA promoter and that lapA is transcriptionally activated by FleQ.FleQ has also been found to be a negative regulator of the expression of genes with a role in exopolysaccharide synthesis in P. fluorescens F113 and P. putida KT2440 that have not been previously identified as regulated by FleQ in any pseudomonad. Among these genes some are related with c-di-GMP turnover, regulation, transport and notably iron homeostasis. Gene ontology analysis showed that genes with similar functions are putatively regulated by FleQ in both species, showing again the functional homology of FleQ proteins. Regarding iron, we have shown binding of FleQ to the pvd promoters, responsible for the synthesis of the major siderophore pyoverdine in F113. Peaks in the regions of these promoters have also been found in the KT2440 genome, although at a lower significance value than in F113 (not shown). Many other iron-responsive genes implicated in iron uptake also showed binding of FleQ in their promoter regions in both species. Expression analysis of several of these genes in F113, KT2440 and theirs fleQ mutant backgrounds, under iron limitation, has shown that deprived of iron these genes are under positive regulation by FleQ in both species. These results determine a novel role for FleQ proteins in pseudomonads, as positive regulators of iron homeostasis.We have also found that FleQ is likely to regulate genes and operons in P. fluorescens F113 and P. putida KT2440 is amrZ. In our ChIP-seq analysis with FleQ, enriched regions have been found upstream of the amrZ gene in both species. Furthermore, gene expression analysis has shown that amrZ is under transcriptional repression by FleQ. It is important to notice that fleQ itself is under strong AmrZ repression both in P. aeruginosa31 and P. fluorescens27. Since AmrZ is also a major regulator of motility and biofilm formation and has been found to negatively regulate iron homeostasis genes in P. aeruginosa29 and P. fluorescens F11328, we decided to compare the AmrZ and the FleQ direct regulons in F113. We have found that 45 genes are putatively regulated by both TFs. Among these genes it is noteworthy that almost every iron-related gene that is directly regulated by AmrZ, is also directly regulated by FleQ. For these iron homeostasis genes, AmrZ acts as a weak repressor28 and FleQ as a weak activator, showing therefore opposing roles. If AmrZ and FleQ interact between them or whether they compete for same regions in the promoter of these genes is currently unknown. In the case of motility and exopolysaccharides genes, most are regulated either by AmrZ or by FleQ although a few of them are overlapping genes. The function of this reciprocal regulation is unclear, although it could work as an oscillator, as indicated in Fig. 30. FleQ in turn has been shown to bind c-di-GMP in P. aeruginosa16 and P. putida17 and it was revealed that this binding determines its transcriptional activity.Other novel gene regulated by FleQ in P. fluorescens strains Pf0-136 and SBW2537. Although these strains and F113 belong to the P. fluorescens complex of species, they have been shown to be different species, belonging to different phylogenetic groups38. In both cases, Pf0-1 and SBW25, more than one hundred genes were shown to be differentially expressed in the wild-type strain compared to the fleQ mutant. These differentially expressed genes belong to different functional classes, showing that in these strains, FleQ is also a global regulator. Although many genes are common with genes reported here to be putatively regulated by FleQ in F113 and P. putida KT2440, iron homeostasis related genes were not found to be regulated by FleQ in Pf-01 or SBW25. However, in the case of SBW25, experiments were performed in iron-sufficient medium (LB) where iron uptake genes are under strong repression by Fur and are not expressed. Furthermore, the level of activation reported in our studies for several of these genes, would have been below the threshold used in both microarrays studies. This indicates that ChIP-seq is valuable in identifying entire regulons of master regulators.Genome wide analysis of the FleQ regulon have been previously performed by microarray hybridization in 18 and it seems to be able to function with different sigma factors: σ54 in the case of most flagellar genes10, σ28 in the case of fliC8, and σ70 in the case of biofilm genes14. In this sense, it has been proposed a role for FleQ in interplay with c-di-GMP and several sigma factors in determining P. putida life-style, switching between flagellar motility and biofilm formation4. On the other hand, FleQ is widely conserved among the pseudomonads and seems to regulate similar genes. The results presented here describe for the first time the implication of FleQ in the regulation of iron homeostasis. Whether FleQ might interact with the subfamily of extracytoplasmic function (ECF) sigma factors in the regulation of iron homeostasis is subject of current investigation. All in all, the results presented in this work allow to conclude that together with AmrZ, FleQ is an important determinant for environmental adaption.FleQ is an atypical EBP. Being a global regulator, it acts both as a transcriptional activator and as a repressor. Its activity depends on the levels of the second messenger c-di-GMP. Conversely to other EBPs, its activation does not depend on phosphorylation by an histidine kinaseP. fluorescens F113 strains were used, a WT strain (F113Rif)39 a fleQ mutant constructed in this work by homologous recombination using pK18mobsacB vector40, a bifA- and a sadC−wspR− strains32. Additionally, three P. putida KT2440 strains were used, a WT strain, which is a plasmid-free derivative of P. putida mt-241, a fleQ mutant26 and a bifA−strain33. F113, KT2440 and derivatives were grown at 28 °C in Luria-Bertani (LB) medium42 for the ChIP-seq experiments, sucrose-asparagine (SA)38 or LB media for swimming with P. fluorescens F113 and P. putida KT2440 and RT-qPCR assays with P. putida KT2440 or CAS medium (3.18 mM Ca(NO3)2, 1 mM MgSO4, 50 mM PIPES, pH adjusted to 6.8, containing 1 mM K2HPO4, 1% (w/v), casamino acids, and 1% (w/v) glycerol) supplemented with 100 µM 2,2’-bipyridyl (low-Fe)43 for RT-qPCR analysis with P. fluorescens F113. Escherichia coli strain DH5α (Gibco-BRL) carrying the appropriate plasmids for each conjugation was grown in LB medium at 37 °C. When solid growth medium was used, 1.5% (w/v) of purified agar was added. Antibiotics were supplemented to maintain or select for plasmids and mutants as follows: ampicillin (Amp) at 100 µg/mL, kanamycin (Km) at 50 µg/mL for P. fluorescens F113 or 25 µg/mL for P. putida KT2440, tetracycline (Tet) at 10 µg/mL for E. coli and 10–25 µg/mL for P. fluorescens F113 and P. putida KT2440.In this study, four P. fluorescens F113 and HAFleQ (5′-ATGTCTTATCCATACGATGTTCCAGATTATGCTTGGCGTGAAACCAAGATT-3′) and FleQR (5′-AAGCTTAATCCTCCGCCTGGTC-3′) for P. putida KT2440. The amplified fragments were cloned into the IPTG-inducible expression vector pVLT3144 to generate the plasmids pBG1998 and pMIR212, respectively.The hemagglutinin peptide YPYDVPDYA (HA) was fused in-frame to the FleQ protein N-terminal domain by PCR using the primer: HAFleQ (5′-ATGTCTTATCCATACGATGTTCCAGATTATGCTTGGCGTGAAACCAAAATTC-3′) and FleQR (5′-TCAATCATCCGCCTGTTCAT-3′) in the case of fleQ mutants of P. fluorescens F113 and P. putida KT2440. Mutant complementation was done by introducing the recombinant plasmid pVLT31 either empty or carrying HA-FleQ into the corresponding mutant strain by triparental mating as reported previously26.The functionality of the HA-FleQ fusion constructs used in the ChIP-seq experiments was validated by the restoration of motility in the fleQ mutants and complemented fleQ mutants was tested by swimming assays. Cells were inoculated in triplicate in SA or LB media on 50 mm diameter plates containing 0.3% (w/v) purified agar by introducing a toothpick with the strain to be analyzed from previous solid cultures for F113 and liquid cultures for KT2440 and incubated at 28 °C. Haloes were observed after 24 h (LB) or 48 h (SA) incubation.Motility of either WT F113 or KT2440, 28. In this experiment, 20 mL of LB-cultures at OD600 of 0.5 from P. fluorescens F113 and derivatives (pBG1998) and P. putida KT2440 and derivatives (pMIR212) were induced for 3 h with 0.1 mM IPTG28. Before immunoprecipitation a sample was prepared to be used as input in order to detect non-specific binding against the IP sample. Samples from four independent cultures per case were inmunoprecipitated and the DNA pooled. Sequencing of DNA samples was carried out by UT Health Science Center at San Antonio Genome Sequencing Facility using Illumina HiSeq. 3000 System single end (50 bp each read).Protein-DNA interaction and binding sites of FleQ were surveyed by Next-Generation Sequencing technology combined with chromatin immunoprecipitation (ChIP). In the ChIP experiment, transcriptions factors were cross-linked to DNA in their native state and immunoprecipitated (IP) following the experimental procedure as detailed previously45, defining a sliding window of 4 nucleotides (nts) with an average Phred quality of 20 and 50 nts as minimum read length to be conserved.In order to remove Illumina adapters and low quality reads, sequences were clipped and filtered with TrimmomaticP. fluorescens F113 or P. putida KT2440 reference genome from GenBank (NC_016830 and NC_002947.4 respectively) was carried out using Bowtie v246. Then, unmapped reads were cleaned with SamTools47 and Picard tools 2.4.148. Reads number from input and IP files were equalized by random subsampling (n = 3) using an own designed Python script. Subsequently, a final alignment was conducted with Bowtie v2. Peak calling was done with MACS 1.4.249 comparing input and IP files and specifying a q-value or false discovery rate (FDR) of 0.01. Peak distribution was visualized in Artemis release 16.0.050 with the purpose of assigning a gene to each peak. Peaks with a fold-enrichment equal or greater than five were selected for P. fluorescens F113 data  at RT and supernatants were discarded. Then, 100 µL of RNAlater  was added to the cell pellets and these conserved at 4 °C.Total RNA from F113, KT2440 and the ® spectrophotometer. RNA integrity was confirmed in 0.8% (w/v) denaturing agarose gels. In addition, genomic DNA contamination in the samples was discarded by PCR  with the primers designed for RT-qPCR experiments . Concentration and quality of the samples was determined using Nanodrop® Reverse Transcriptase (Invitrogen). Then, qPCR reactions of the cDNA synthesized were carried out in quadruplicate for each gene, using FastStart Universal SYBR Green Master Rox (Roche).Complementary DNA (cDNA) synthesis by reverse transcription (RT-PCR) was performed using Superscript IV16S rRNA expression as housekeeping and relativized to F113 or KT2440 WT following the 2−ΔΔCt method54.For both strains, two biological replicates were considered and gene expression was calculated using threshold cycle (Ct) values. Data was normalized by using 55 and VennDiagram56 package in R software57 was used in the representation of the plot for genomic distribution of peaks and Venn diagram respectively. GraphPad Prism version 7.00 for Windows  was used in the statistical analysis and representation of RT-qPCR data, the comparison was done using multiple t-test for independent samples (p < 0.05) with Bonferroni-Dune method; and for the representation of the pie charts.R CommanderSupplementary Information


Primary production on Earth is dependent on autotrophic carbon fixation, which leads to the incorporation of carbon dioxide into biomass. Multiple metabolic pathways have been described for autotrophic carbon fixation, but most autotrophic organisms were assumed to have the genes for only one of these pathways. Our finding of a cultivable bacterium with two carbon fixation pathways in its genome, the rTCA and the CBB cycle, opens the possibility to study the potential benefits of having these two pathways and the interplay between them. Additionally, this will allow the investigation of the unusual and potentially very efficient mechanism of electron flow that could drive the rTCA cycle in these autotrophs. Such studies will deepen our understanding of carbon fixation pathways and could provide new avenues for optimizing carbon fixation in biotechnological applications. Riftia pachyptila, the fastest-growing invertebrate. To date, the coexistence of these two carbon fixation pathways had not been found in a cultured bacterium and could thus not be studied in detail. Moreover, it was not clear if these two pathways were encoded in the same symbiont individual, or if two symbiont populations, each with one of the pathways, coexisted within tubeworms. With comparative genomics, we show that Thioflavicoccus mobilis, a cultured, free-living gammaproteobacterial sulfur oxidizer, possesses the genes for both carbon fixation pathways. Here, we also show that both the CBB and rTCA pathways are likely encoded in the genome of the sulfur-oxidizing symbiont of the tubeworm Escarpia laminata from deep-sea asphalt volcanoes in the Gulf of Mexico. Finally, we provide genomic and transcriptomic data suggesting a potential electron flow toward the rTCA cycle carboxylase 2-oxoglutarate:ferredoxin oxidoreductase, via a rare variant of NADH dehydrogenase/heterodisulfide reductase in the E. laminata symbiont. This electron-bifurcating complex, together with NAD(P)+ transhydrogenase and Na+ translocating Rnf membrane complexes, may improve the efficiency of the rTCA cycle in both the symbiotic and the free-living sulfur oxidizer.Very few bacteria are able to fix carbon via both the reverse tricarboxylic acid (rTCA) and the Calvin-Benson-Bassham (CBB) cycles, such as symbiotic, sulfur-oxidizing bacteria that are the sole carbon source for the marine tubeworm IMPORTANCE Primary production on Earth is dependent on autotrophic carbon fixation, which leads to the incorporation of carbon dioxide into biomass. Multiple metabolic pathways have been described for autotrophic carbon fixation, but most autotrophic organisms were assumed to have the genes for only one of these pathways. Our finding of a cultivable bacterium with two carbon fixation pathways in its genome, the rTCA and the CBB cycle, opens the possibility to study the potential benefits of having these two pathways and the interplay between them. Additionally, this will allow the investigation of the unusual and potentially very efficient mechanism of electron flow that could drive the rTCA cycle in these autotrophs. Such studies will deepen our understanding of carbon fixation pathways and could provide new avenues for optimizing carbon fixation in biotechnological applications. Riftia, Escarpia, Tevnia, and Lamellibrachia, which have and express both the oxygen-sensitive rTCA and the oxygen-tolerant CBB cycle  and transcriptome of the symbiont from the tubeworm aclAB genes), 2-oxoglutarate:ferredoxin oxidoreductase , and a putative fumarate reductase  from a subsurface aquifer (Gammaproteobacterium RIFOXYD12_FULL_61_37) , were aL_61_37)  Fig. 1)aclAB genmic tree . The tub or both , Note 1.10.1128/mSphere.00394-18.1TEXT S1Text S1, DOCX file, 0.01 MB.Supplemental methods, notes, and references. Download Copyright © 2019 Rubin-Blum et al.2019Rubin-Blum et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00394-18.2FIG S1aclA sequences from Gammaproteobacteria. All positions containing gaps and missing data were eliminated. There were a total of 707 positions in the final data set. The gray box frames tubeworm sequences; purple color marks Chromatiaceae sequences. * marks the sequence of Magnetococcus marinus, in which the function of the ATP citrate lyase was biochemically tested. Download FIG S1, EPS file, 2.3 MB.Phylogeny of ATP-citrate lyase large subunit (AclA). The evolutionary history was inferred by using the maximum likelihood method based on the Le-Gascuel 2008 model . A discrete gamma distribution was used to model evolutionary rate differences among sites . The rate variation model allowed for some sites to be evolutionarily invariable . The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. We used 46 amino acid sequences for the analysis, including all known Copyright © 2019 Rubin-Blum et al.2019Rubin-Blum et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. laminata symbiont was similar to that of single-copy marker genes . Phage genes are shown as an example of a gene cluster that was present only in a subpopulation of the symbionts. The very low expression values for the strain-specific phage cluster are not shown, as the expression values for strain-specific genes cannot be measured accurately. Download Table S1, DOCX file, 0.02 MB.Uniform sequencing read coverage of the rTCA and Calvin cycle gene clusters and their expression values. All values are normalized to that of the Copyright © 2019 Rubin-Blum et al.2019Rubin-Blum et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00394-18.10TABLE S2Table S2, DOCX file, 0.01 MB.Accession numbers of genomes used in comparative analyses. Download Copyright © 2019 Rubin-Blum et al.2019Rubin-Blum et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. laminata tubeworm symbionts revealed high expression levels of both the rTCA and the CBB cycle genes (Riftia symbiont (the metabolism of the symbionts from these two tubeworms is highly similar)  , 7. The similar) .T. mobilis, and the gammaproteobacterial MAG from a subsurface aquifer, there was a considerable level of conservation of the rTCA gene clusters, at the sequence and synteny levels , idh (isocitrate dehydrogenase), and mdh . The other rTCA-specific genes korABCD (four-subunit OGOR) and tfrAB (putative thiol:fumarate reductase) were also present in the rTCA gene cluster. Similar to the ACL, the four-subunit OGOR and the thiol:fumarate reductase are very rare among Gammaproteobacteria and were probably acquired via a single horizontal gene transfer event from a distant bacterial clade (korAB genes), more common than the four-subunit enzyme among gammaproteobacterial autotrophs, yet absent in T. mobilis, was located elsewhere in the genome of the E. laminata symbiont. The korAB genes were colocalized with genes that encode other well-expressed TCA cycle enzymes (gltA) gene, which could indicate its use in the catabolic oxidative TCA cycle. Alternatively, strong expression of citrate synthase could also indicate autotrophic CO2 fixation via the recently discovered ACL-independent reverse oxidative TCA (roTCA) cycle  cycle , 22.10.1128/mSphere.00394-18.3FIG S2Chromatiaceae sequences. Biochemically characterized archaeal sequences were not included in the tree to be able to present the clade of interest in high resolution. Download FIG S2, EPS file, 2.4 MB.Phylogeny of the alpha subunit (KorA) of the 4-subunit 2-oxoglutarate:ferredoxin oxidoreductase . The evolutionary history was inferred using the maximum likelihood method based on the Le-Gascuel 2008 model . A discrete gamma distribution was used to model evolutionary rate differences among sites . The rate variation model allowed for some sites to be evolutionarily invariable . The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The gray box frames tubeworm sequences; purple color marks Copyright © 2019 Rubin-Blum et al.2019Rubin-Blum et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00394-18.4FIG S3Chromatiaceae sequences. The distant TfrA sequence from Methanobacterium thermoautotrophicum was not included in the tree to present the clade of interest in high resolution. Download FIG S3, EPS file, 2.1 MB.Phylogeny of the alpha subunit (TfrA) of the thiol:fumarate reductase . The evolutionary history was inferred using the maximum likelihood method based on the Le-Gascuel 2008 model . A discrete gamma distribution was used to model evolutionary rate differences among sites . The rate variation model allowed for some sites to be evolutionarily invariable . The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The gray box frames tubeworm sequences; purple color marks Copyright © 2019 Rubin-Blum et al.2019Rubin-Blum et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00394-18.5FIG S4Escarpia laminata tubeworm symbionts. (B) The TCA cycle reconstruction. Each box is named after the respective protein. Frame color corresponds to a gene cluster, and box color corresponds to the gene color code in panel A. FumB is not colored as the standalone fumB gene is located elsewhere in the genome. OAA, oxaloacetate. Download FIG S4, EPS file, 2.5 MB.Genome-based reconstruction of the TCA cycle in tubeworm symbionts. (A) Expression profile of both gene clusters that encode the TCA cycle in Copyright © 2019 Rubin-Blum et al.2019Rubin-Blum et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the flxABCD-hdrABC genes 2+/1+ centers, respectively. Download FIG S5, EPS file, 2.2 MB.Proposed function of the putative HdrABC-FlxABCD-KorABCD-TfrAB complex. Electrons from NADH oxidation are transferred to HdrA, which may bifurcate them to KorABCD  and to TfrAB . Squares and hexagons mark the estimated presence of [4Fe-4S]Copyright © 2019 Rubin-Blum et al.2019Rubin-Blum et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the T. mobilis, in which the genes for the CBB and rTCA cycles coexist. Such studies would reveal if these pathways are expressed under different physicochemical conditions and potentially allow the biotechnological optimization of efficiency and yield in production processes that rely on autotrophic carbon fixers. To our knowledge, the use of organisms with multiple carbon fixation pathways has not been used as a design principle for these applications.Until now, the only bacteria known to possess both the CBB and rTCA pathways were sulfur-oxidizing, tubeworm symbionts, and possibly also large sulfur bacteria, all of which are currently not amenable to cultivation-based studies. Experimental studies are now feasible in the cultivable de novo-assembled genomes of Escarpia laminata symbionts (estimated completeness 99.5%), were used for genomic comparison . Twenty-three marker proteins that are universally conserved across the bacterial domain were extracted from genomes using the AMPHORA2 pipeline  from the 2-subunit 2-oxoglutarate:ferredoxin oxidoreductase . The evolutionary history was inferred by using the maximum likelihood method based on the Le-Gascuel 2008 model . A discrete gamma distribution was used to model evolutionary rate differences among sites . The rate variation model allowed for some sites to be evolutionarily invariable . The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The gray box frames tubeworm sequences; purple color marks Copyright © 2019 Rubin-Blum et al.2019Rubin-Blum et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00394-18.8FIG S7Chromatiaceae sequences. *, the function of the HdrABC-FlxABCD was tested in the deltaproteobacterium Desulfovibrio vulgaris. Download FIG S7, EPS file, 2.5 MB.Phylogeny of the alpha subunit (HdrA) from the NADH dehydrogenase/heterodisulfide reductase (HdrABC-FlxABCD) electron-bifurcating complex, colocalized with the 4-subunit OGOR . The evolutionary history was inferred by using the maximum likelihood method based on the Le-Gascuel 2008 model . A discrete gamma distribution was used to model evolutionary rate differences among sites . The rate variation model allowed for some sites to be evolutionarily invariable . The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The gray box frames tubeworm sequences; purple color marks Copyright © 2019 Rubin-Blum et al.2019Rubin-Blum et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


Apolygus lucorum Meyer-Dür, 1843, an omnivorous species that feeds on plants and animals, has become a major pest in China as production of Bt-cotton has grown to such a large scale. Its omnivory is likely to be critical for its success, but the digestive mechanism(s) underlying processing and adsorption of such diverse foods is relatively unknown. Here, we examined the activities of digestive enzymes of A. lucorum in the salivary gland complex and midgut and the effect of sex, age, and food source on these activities. Amylase and protease were present in the salivary gland complex and the midgut, but were higher in the salivary gland complex. Trypsin-like enzyme was also present in both organs, but chymotrypsin-like enzyme was present only in the midgut. Sex, age, and food source affected the activities of these digestive enzymes. In general, the activities of these enzymes peaked at 10 d after emergence, and amylase and protease activities were higher in female adults than in males. Of the food sources tested, green bean pods (Gb) induced the highest amylase activity, whereas Helicoverpa armigera Hübner, 1809 eggs (He) and a mixture of Gb and He induced higher activities of the trypsin-like and chymotrypsin-like enzymes. The results from food switching experiments confirmed that amylase activity could be induced by plant sources, and animal sources induced protease activity. Thus, the types and activities of digestive enzymes in A. lucorum provide the physiological basis of the pest’s omnivory.The mirid bug  Apolygus lucorum Meyer-Dür, 1843 (Hemiptera: Miridae) has become a major pest of cotton in conjunction with the large-scale adoption of Bt-cotton  in recent decades in China , nymphs of Aphis gossypii Glover (Hemiptera: Aphididae) and Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae)  . Previouollicles .Poecilocapsus lineatus F. (Heteroptera) lacks detectable digestive proteases  and Lygushesperus Knight have amylases and trypsin-like enzymes in their salivary gland complexes  h  h .A. lucorum in both sexes at different ages, we excised the salivary gland complex and midgut separately from 1-, 5-, 10-, 15-, 20-, and 25-d-old male and female adults from the laboratory colony.For the assays of the digestive enzymes of A. lucorum originate in the salivary gland complex rather than the midgut, and because enzyme activities were greater in female adults than in male adults, the salivary gland complex from female adults was used in experiments on different foods and on food switching. For comparing the activities of digestive enzymes of A. lucorum reared on different foods, three types of food sources were used: 1) green bean pods (Gb) for the plant source, 2) a plant and an animal source together, green bean pods and H. armigera eggs (GbHe), and 3) the animal source alone, H. armigera eggs (He). Female adults that had fed on the Gb and GbHe diet at 1, 5, 10, 15, 20, and 25 d old and on the He diet at 1, 5, and 10 d old  were collected for dissecting salivary gland complex for digestive enzymes assay.Because the digestive enzymes in A. lucorum, three food sources  were used. Five-day-old female adults that were reared on Gb, GbHe, or He since they had hatched from eggs were used for the experiments. Six treatments were designed: 1) Gb diet, then transfer to GbHe (Gb-to-GbHe); 2) Gb diet, then transfer to He (Gb-to-He); 3) GbHe diet, then transfer to Gb (GbHe-to-Gb); 4) GbHe diet, then transfer to He (GbHe-to-He); 5) He diet, then transfer to Gb (He-to-Gb); 6) He diet, then transfer to GbHe (He-to-GbHe). Those kept on the original food were used as controls. Female adults were collected at 1, 3, 5, 7, and 10 d after the transfer in the first four treatments for excising salivary gland complex; in the other two treatments, female adults were collected 1, 3, and 7 d after the transfer because the average longevity of female adults fed on the He diet was 10 d.To test the effect of food source switching on the activities of digestive enzymes in A. lucorum were starved for 12 h before dissection. The insects were dissected in ice-cold phosphate-buffered saline  using a stereomicroscope. The salivary gland complex, including all lobes, accessory glands, and tubules, was exposed by removing the prothorax from the abdomen with fine forceps. The midgut was exposed by holding the body with the forceps and pulling the last three or four segments of the abdomen away from the rest of the abdomen with another pair of forceps. The salivary glands and midguts from A. lucorum were collected separately, then put in a cold homogenizer with PBS. Tissues were homogenized and then transferred to a 1.5-ml centrifuge tube and centrifuged at 12,000 rpm for 15 min at 4°C. The resulting supernatants were maintained at −80°C for further use. At least three biological replicates were assayed as triplicate technical replicates for each treatment, and each biological replicate was collected from 50 adults.Enzyme samples were prepared using the method of The protein concentrations of all of the enzyme samples were determined using the Bradford method with bovine serum albumin as a reference standard according to the manufacturer’s instructions .Amylase activity was assayed using the dinitrosalicylic acid (DNS) procedure  with 1% Nα-benzoyl-l-arginine-p-nitroanilide  in 1 ml of dimethyl methyl sulfoxide before adding 49 ml of universal buffer (pH 7.5) to obtain a final BApNA concentration of 1 mg/ml. Ten microliters of enzyme extract was mixed with 40 μl of universal buffer in a microtiter plate, and the mixture was incubated at 30°C for 20 min before 100 μl substrate was added. Absorbance was measured at 405 nm. A no-enzyme control was included to correct for endogenous hydrolysis of substrate. One unit of enzyme (U) is defined as the amount that hydrolyzes 1 μmol of substrate per minute. Mean enzyme activity were calculated from triplicate readings. The experiment was repeated with different enzyme preparations.The activity of trypsin-like enzymes was assayed as previously described  with sliN-succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide  in 1 ml of dimethylformamide before adding 24 ml of universal buffer to obtain a final SAAPPpNA concentration of 1 mg/ml. Five microliters of enzyme sample was mixed with 90 μl of universal buffer in a microtiter plate, and the mixture was incubated at 30°C for 20 min before 45 μl substrate was added. Absorbance was measured at 405 nm. Chymotrypsin-like enzyme activity was calculated as done for trypsin-like enzyme activity. One unit of enzyme (U) was defined as the amount that hydrolyzes 1 μmol of substrate per minute.Chymotrypsin-like enzyme activity was assayed as previously described  with sliData were subjected to an analysis of variance (ANOVA), and the means were compared by a least square difference test (LSD). Statistical analyses were performed using the software Statistics Analysis System V8.A. lucorum adults secreted amylase in the salivary gland complex and midgut at different ages  in the midgut of 10-d-old females. Chymotrypsin-like enzyme activities in the midgut of female and male adult fluctuated with increasing age. The results showed that enzyme activities in the midgut were higher in females than in males of all ages except 20-d-old adults.Chymotrypsin-like enzyme activities measured in extracts of the salivary gland complex and midgut of A. lucorum had higher amylase activities than those fed on He and GbHe. Protease activity (trypsin-like and chymotrypsin-like enzymes) in adults fed on He and GbHe was higher than in the Gb-fed adult.The activities of amylase, trypsin-like and chymotrypsin-like enzymes differed among food sources . Gb-fed Apolyguslucorum fed on Gb had higher amylase activity than those fed on He and GbHe . In 15-, 20-, and 25-d-old female adults, amylase activity was significantly higher with the Gb diet than with GbHe .and GbHe . For theApolyguslucorum on the Gb diet had lower trypsin-like enzyme activity than on the He or GbHe diet . In insects 5 d old, trypsin-like enzyme activity with the He diet was significantly higher than with Gb, but did not differ from GbHe . In insects 10 d old, trypsin-like enzyme activity with GbHe was significantly higher than with Gb, but did not differ from He . In 15-, 20-, and 25-d-old adults, trypsin-like enzyme activities did not differ significantly between Gb and GbHe .bHe diet . The higApolyguslucorum fed on He or GbHe had high activities of chymotrypsin-like enzyme but none on Gb treatment , but there was no significant difference at 1 d of age .reatment . With inA. lucorum adults were transferred from Gb to He or GbHe, amylase activities tended to decline with increasing time after the switch .The activities of amylase changed distinctly with food switching . When A.e switch . The decF2,8 = 4.69, P = 0.045; for 10 d after: F2,11 = 9.41, P = 0.004).Compared with the activity for the GbHe controls, amylase activity increased in the GbHe-to-Gb test, but did not change in the GbHe-to-He test . With inF2,7 = 4.99, P = 0.045; for 7 d after: F2,7 = 4.24, P = 0.042).Amylase activities increased starting by 1 d after the transfer from He to either Gb or GbHe . At 3 anA. lucorum on the Gb-to-GbHe and Gb-to-He treatments increased with transfer time compared with the Gb control . At 7 d after the transfer, the activities for the Gb-to-GbHe and Gb-to-He treatments were 864.02 ± 148.75 and 777.01 ± 101.01 U/mg protein, 2.12 and 1.91 times higher than for the Gb control.In general, trypsin-like enzyme activities in  control . SignifiF2,8 = 8.52, P = 0.010; for 5 d after: F2,8 = 15.03, P = 0.002; for 7 d after: F2,8 = 14.54, P = 0.002; for 10 d after: F2,9 = 13.77, P = 0.002).Trypsin-like enzyme activities increased in the GbHe-to-He treatment, but decreased in GbHe-to-Gb treatment compared with the GbHe control . The actF2,7 = 16.65, P = 0.002).For the transfer from He to the other two food items, trypsin-like enzyme activities increased in the He-to-GbHe test but decreased in the He-to-Gb . The actP > 0.05).Chymotrypsin-like enzyme activity was not detected in insects feeding on Gb until 2 d after the transfer from Gb to GbHe or He . ChymotrF2,9 = 4.08, P = 0.046; for 7 d after: F2,8 = 5.82, P = 0.028). At 10 d after the transfer, the substrate for the chymotrypsin-like enzyme was not hydrolyzed by the enzyme sample from the bugs in the GbHe-to-Gb test. No significant difference was found between the GbHe and GbHe-to-He tests at 10 d after transfer .Chymotrypsin-like enzyme activities increased in the GbHe-to-He treatment but decreased in the GbHe-to-Gb compared with the GbHe control . The actA. lucorum were transferred from He to the other two food items, chymotrypsin-like enzyme activity increased in the He-to-GbHe test but decreased in the He-to-Gb . At 7 d after transfer, no substrate was hydrolyzed by the enzyme sample from the He-to-Gb treatment.When He-to-Gb . HoweverA. lucorum. This species had amylase activity, especially in the salivary gland complex, which could indicate that it may extraorally digest and liquefy plant food sources. Amylase activity in the salivary glands and midguts of other phytophagous heteropterans has also been reported , indicating that this species is not a strict phytophage.The composition and activity of digestive enzymes can reflect the consumer’s ability to use plant or animal food sources . Here, wreported . For insactivity . AmylaseApolyguslucorum had strong protease activity, especially in the salivary gland complex, which indicates it can use extraoral digestion to use structural proteins in animal food sources. L. hesperus. Variations in protease activity were also found in the predator Podisus maculiventris (Say) (Hemiptera: Pentatomidae) (P.lineatus (Heteroptera: Miridae).tomidae) . Cohen aA. lucorum further affirmed the ability of this mirid to extraorally digest proteins from animal food sources. A previous study showed strong trypsin-like enzyme activity in the assassin bug Zelus renardii Kolenati (Hemiptera: Reduviidae)  . We alsos insect .A. lucorum, amylase and trypsin-like enzyme activities in the salivary gland complex of females at 10 d of age were 0.97 ± 0.10 U/mg and 507.61 ± 39.82 U/mg, respectively. In predatory bug, Podisus nigrispinus, amylase and cathepsin (the main protease in this bug) activities were 0.02 ± 0.004 U/mg and 3800 ± 300 U/mg  of female adults tested were found in the salivary gland complex and midgut of A. lucorum. These may indicate that the digestive enzymes appear to have crucial roles in reproduction. During the period, from preoviposition to the peak of oviposition of A. lucorum, high nutrient levels are required  activity was higher than in adults fed only plants. High protease activity in the adults fed on the combination food and on the animal food indicated that this mirid could also use animal material for nutrition. Food sources also affect the activity of digestive enzymes of other insects. Digestive enzymes of Gryllus bimaculatus (Orthoptera: Gryllidae) released in response to nutrients in the food  (Lepidoptera: Noctuidae) adjust amylase activity for carbohydrate ingestion, and indeed, amylase activity is significantly higher in fed carbohydrate larvae than in unfed larvae (the food . Sugars d larvae .A. lucorum by 5 or 7 d after the transfer to a different diet. Thus, within a certain time, the activities of the digestive enzymes in A. lucorum can change in response to changes in nutrient conditions of the diet. Other insects can respond similarly (B. tabaci (P. maculiventris (Results of the food switching test showed that amylase and protease activities changed significantly in the salivary gland complex of imilarly . For exa. tabaci . Differeiventris .A. lucorum indicates that this species can be both phytophagous and zoophagous. The activities of these enzymes are affected by sex, age, and food source. Such findings reveal the relative levels of phytophagy and zoophagy in both sexes of A. lucorum at different ages. In addition, the results of this study will be useful for laboratory rearing and for further elucidating the feeding mechanism of A. lucorum.In summary, the presence of amylase and protease in the salivary gland complex and midgut of


Actinoplanes sp. SE50/110 is a natural producer of acarbose. It has been extensively studied in the last decades, which has led to the comprehensive analysis of the whole genome, transcriptome and proteome. First genetic and microbial techniques have been successfully established allowing targeted genome editing by CRISPR/Cas9 and conjugal transfer. Still, a suitable system for the overexpression of singular genes does not exist for Actinoplanes sp. SE50/110. Here, we discuss, test and analyze different strategies by the example of the acarbose biosynthesis gene acbC.gusA gene and glucuronidase assays as well as reverse transcription quantitative PCR (RT-qPCR). Additionally, we mapped transcription starts and identified the promoter sequence motifs by 5′-RNAseq experiments. Promoters with medium to strong expression were included into the pSET152-system, leading to an overexpression of the acbC gene. AcbC catalyzes the first step of acarbose biosynthesis and connects primary to secondary metabolism. By overexpression, the acarbose formation was not enhanced, but slightly reduced in case of strongest overexpression. We assume either disturbance of substrate channeling or a negative feed-back inhibition by one of the intermediates, which accumulates in the acbC-overexpression mutant. According to LC–MS-analysis, we conclude, that this intermediate is valienol-7P. This points to a bottleneck in later steps of acarbose biosynthesis.The integrative φC31-based vector pSET152 was chosen for the development of an expression system, as for the replicative pSG5-based vector pKC1139 unwanted vector integration by homologous recombination was observed. Since simple gene duplication by pSET152 integration under control of native promoters appeared to be insufficient for overexpression, a promoter screening experiment was carried out. We analyzed promoter strengths of five native and seven heterologous promoters using transcriptional fusion with the Actinoplanes sp. SE50/110 is an important step for future metabolic engineering. This system will help altering transcript amounts of singular genes, that can be used to unclench metabolic bottlenecks and to redirect metabolic resources. Furthermore, an essential tool is provided, that can be transferred to other subspecies of Actinoplanes and industrially relevant derivatives.Development of an overexpression system for The online version of this article (10.1186/s12934-019-1162-5) contains supplementary material, which is available to authorized users. Actinoplanes sp. SE50/110 (ATCC 31044), is a natural derivative of SE50. It was isolated from a soil sample during a screening program by the Bayer AG in 1970 as natural producer of an α-glucosidase inhibitor  , which might catalyze such kind of recombination process. Furthermore, no homolog was found in the genome of A. teichomyceticus (not shown), which might explain, why such process has not been reported for this species before.Homologous recombination is a common process in Actinobacteria: in various ces ssp. –29. In cyceticus . In thisyceticus  lead to A. teichomyceticus and although high vector stability has already been shown for the empty vector in SE50/110, the replicative pSG5-based vector pKC1139 is not suitable as expression vector in Actinoplanes sp. SE50/110, as vector integration by homologous replication seems to be a favored process, putatively due to the metabolic costs of vector replication. A pSG5-based expression system might be implemented in future by deletion of the recombinase gene recA.In contrast to Actinoplanes sp. SE50/110.Consequently, integrative vector systems were tested as vehicles for the expression of homologous genes in Actinoplanes sp. SE50/110 . Exconjugants were grown in maltose minimal medium displaying normal growing and acarbose producing phenotypes compared to the empty vector control Actinoplanes sp. SE50/110 [pSET152] and the wild-type (data not shown). Interestingly, RT-qPCR experiments showed, that no duplication of transcript amount was achieved for each of these constructs compared to the empty vector control , which carries the φC31-integration site, by influencing of the transcription of the genes in direct vicinity to the integration site, or by integration of the vector into secondary (pseudo)-integration sites.This might be caused by nonspecific effects, like f. e. by deletion of the gene ACSP50_6589 is annotated as hypothetical pirin-homologue  Fig. . The comacb gene cluster is disrupted by unintended vector integration  gave the statistically most valid result for the formula C7H12O8P, which corresponds to valienol-7-phosphate . However, although suitable as integration tool, genetic engineering by CRISPR/Cas9 is cost- and time-consuming. For fast screening of overexpression mutants, f. e. for the detection of intermediate products, the pSET152-expression system is still the best tool, as it allows easy screening of several promoters in parallel.By knowledge of the strength of several promoters in the host Actinoplanes sp. SE50/110 (ATCC 31044) was grown in the complex medium NBS  and mixed 2:3 with sterile 86% (v/v) glycerol. Glycerol stocks are stored at − 80 °C. A spore solution was prepared from solid culture, like described by Wolf et al. . Data S3. Results of promoter screening by GUS-assay. Data S4. Determination of transcription start sites of heterologous promoters in Actinoplanes sp. SE50/110 by 5‘-end specific transcriptome sequencing. Data S5. Growth and acarbose formation of pSET152-based acbC-overexpression mutants. Data S6. Smart formula analysis of the isotopic pattern of mass = 255.03 [M−H+].


The mean pain scale values with 23- and 25-gauge needles were 2.1 ± 1.3 and 1.6 ± 1.3, respectively (p = 0.135). There were no significant complications in either group. In conclusion, both 23- and 25-gauge needles are useful in LBC because cytological adequacy and complications were not statistically different with both sizes of the needles.In ultrasound (US)-guided fine-needle aspiration (FNA) of solid thyroid nodules (STN) using liquid-based cytology (LBC), the most appropriate needle size for LBC remains unclear. This study compared the cytological adequacy and complications associated with using 23- and 25-gauge needles in US-guided FNA of STNs using LBC. US-guided FNA was performed in consecutive patients by one radiologist to diagnose STNs ≥ 5 mm in the largest diameter. The one-sampling technique through a single needle puncture and multiple to-and-fro needle motions was used in each patient. The 23- and 25-guage needles were used consecutively each day. After FNA, the pain and complications experienced by each patient were investigated by a nurse, who was blinded to the information of needle gauge used. A cytopathologist retrospectively analyzed the cytological adequacy and cellularity of the cases. Of the 99 STNs, eight (8.1%) exhibited inadequate cytology (4 each with 23- and 25-gauge needles). The rate of cytological adequacy was not statistically different between the groups ( For cytological diagnosis of US-guided FNA of thyroid nodules, conventional smears, which are prepared by coating the aspirated material evenly onto multiple microscope slides, have been used for a long time2. Liquid-based cytology (LBC) is rapidly replacing conventional smears since its introduction in 19966. LBC employs advanced concentration techniques to yield a single slide that contains most, or all, of the FNA material4. Numerous factors can influence the cytological adequacy of US-guided FNA of thyroid nodules using LBC: the solidity or vascularity of the thyroid nodule, sampling technique used (aspiration and capillary acquisition) and number of samples taken (single versus multiple), number of needle passes, use of aspiration devices, and gauge of the needle used11. In clinical practice, needles between 21 to 27 gauge are usually selected according to the institution and physician11. While one previous study demonstrated that when solution-based samples are used, larger (21-gauge) needles provide more diagnostic specimen7, another study demonstrated that there was no significant difference between 21-gauge and 23-gauge needles11. Hence, the most appropriate needle size to be used for LBC remains unclear. This study aimed at comparing the cytological adequacy, pain scale, and other complications associated with using 23- and 25-gauge needles in US-guided FNA of thyroid nodules using LBC.Ultrasonography (US)-guided fine-needle aspiration (FNA) is a cost-effective modality used as the mainstay for initial evaluation of thyroid nodulesThis prospective study was approved by the Busan Paik Hospital Institutional Review Board. All research was performed in accordance with relevant guidelines, and informed written consent was obtained from all patients before the US-guided FNA was performed. Between January and April 2018, one radiologist, with seven years of experience in US-guided FNA of thyroid nodules, performed US-guided FNA in 120 consecutive patients  to diagnose thyroid nodules that were ≥5 mm in the largest diameter . Among these, purely calcified thyroid nodules (n = 5), thyroid cysts (n = 8), and partially cystic thyroid nodules (n = 8) were excluded. Ninety-nine patients  with solid thyroid nodules  were included.12. To obtain adequate specimens, the mixed sampling technique  was used12. Aspirated material was collected in CytoLytTM solution (Hologic-Cytyc Co.) as follows: after separating the plastic syringe and needle, the contents within the syringe were injected into the LBC bottle, and then, the contents within the needle were flushed into the LBC bottle.One radiologist performed US-guided FNA in all patients under the guidance of a high-resolution US modality  equipped with a 5- to 12-MHz linear-array transducer. The operator used a conventional 23-gauge (2.5 cm in length) or a 25-gauge (3.8 cm in length) needle attached to an empty 3-mL plastic syringe . The nurse recorded the pain score mentioned by the patients.TM solution containing the aspirates was centrifuged, the obtained pellet was transferred to the PreservCytTM (Hologic-Cytyc Co.) solution and allowed to rest for 15 minutes. In each case, one Papanicolaou-stained slide was prepared by using ThinPrep 5000 (Hologic-Cytyc Co.).After the CytoLyt14. Specimens that contained abundant colloid or numerous inflammatory cells were classified as adequate, even though six groups of follicular cells were not identified. On the contrary, specimens that only consisted of cystic contents were considered inadequate. To determine the cellularity, the numbers of cell clusters ≥ 10 well-visualized follicular cells were counted using a ×100 microscope in the highest cellular area. The cytopathologist classified the degree of cellularity as follows: high (more than 10 clusters), intermediate (5 to 10 clusters), and low (less than 5 clusters) cellularity. Cytomorphologic features were reviewed and categorized using the Bethesda classification.All cytological slides were retrospectively analyzed by a cytopathologist with 16 years of experience in cytological analysis of thyroid FNA, with blinded to the data on the needle size. Specimen adequacy was determined according to the Bethesda System for Reporting Thyroid Cytopathology: six groups of ten well-visualized follicular cells was the minimal criteria2 test, and those for small cell values using the Fisher’s exact test. All statistical analyses were performed with a statistical software , and P values < 0.05 were considered statistically significant.The data was tested for normal distribution using the Kolmogorov-Smirnov test. Age at the time of diagnosis, and the size of nodule were expressed as the mean ± SD. The cases were divided into two groups based on the size of needle used (23-gauge needle group versus 25-gauge needle group). Mean differences in age and size of nodule between the two groups were compared using the independent t-test. Group comparisons of categorical variables were performed using the χThe study design was approved by the appropriate ethics review boards (IRB 18-0135), and informed written consent was obtained from all patients before the US-guided FNA was performed.Consent for publication was not obtained from the patient owing to patient anonymity and no patient details within the manuscript. All the authors have taken public responsibility for the manuscript content and publication.p < 0.0001). However, there were no significant between-group differences in the patient age, gender, and location of STNs (p > 0.05).The mean of the largest diameters of the 99 STNs was 14.4 ± 10.5 mm . The locations of the STNs included the right lobe (n = 50), left lobe (n = 48), and isthmus (n = 1). US-guided FNA was performed using 23-gauge needle for 49 (49.5%) STNs and 25-gauge needles for 50 (50.5%) STNs. The demographic characteristics of the two groups are presented in Table p = 0.631). The 25-gauge needle group showed a higher prevalence in the high cellularity class; the 23-gauge needle group showed a higher prevalence in the intermediate cellularity class; and both the groups had similar prevalence in the low cellularity class. The differences in the cytological cellularity between the groups were not statistically significant (p = 0.185). Among the 99 STNs, there were Bethesda category I (n = 8), II (n = 38), III (n = 25), IV (n = 6), V (n = 11), and VI (n = 11). Bethesda category II STNs were most common in both groups. There was no significant difference in the Bethesda classification between the two groups (p = 0.711).Of the 99 STNs, eight (8.1%) showed inadequate cytology: six revealed no follicular cells, whereas two showed at least one follicular cluster with 20 to 60 follicular cells. The cytological adequacy and cellularity of the two groups are compared in Table p = 0.135). Scores of 1 or 2 were predominant, and no case reported a score more than 8. Intrathyroidal hemorrhage and other complications were not found in any case.The pain scale scores of the two groups are compared in Table 15. However, for LBC, the relationship between the needle gauge used and the adequacy of the FNA specimen is still unclear. Although larger needles provide more specimen for diagnosis7, the difference in cytological adequacies between 21-gauge and 23-gauge needle groups is not significant11. Furthermore, the number of studies available is small. In the present study, we attempted to compare the difference in cytological adequacy of US-guided FNA of STNs for LBC between 23- and 25-gauge needles. There was no statistically significant difference. This result was similar to that of our previous study that compared 21-gauge and 23-gauge needles11. Moreover, the rates of cytological adequacy when using either 23- or 25-gauge needles were high (91.8% and 92%).Thus, we believe that the use of needles between 21- and 25-gauge does not affect the cytological adequacy of US-guided FNA of STNs for LBC.In US-guided FNA of thyroid nodules for conventional smears, thinner needles are associated with better cytological adequacy than larger needles7. Although this difference was not statistically significant, the data trended toward suggesting that the use of 21-gauge needles for FNA increased the specimen cellularity (p = 0.08)7. Other investigators also reported that FNAs of thyroid nodules for LBC performed with 21-gauge needles led to more cellular specimens than those performed with 25-gauge needles16. However, the increased cellularity from the biopsies performed with the 21-gauge needles did not increase the sufficiency rate16. In our previous study, there was no significant difference in the cytological cellularity between 21- and 23-gauge needle groups. However, the 23-gauge needle group showed higher cellularity when compared with the 21-gauge needle group11. Likewise, the present study did not show a significant difference in the cytological cellularity between the 23- and 25-gauge needle groups, and the 25-gauge needle group showed higher cellularity when compared with the 23-gauge needle group. The reason for the disparity between our studies and others is not clear. We did not compare the difference in cytological cellularity between using 21- and 25-gauge needles.In a previous study, samples collected using 21-gauge needles in US-guided FNA of thyroid nodules for LBC produced an average cellularity of 2.18, whereas those collected using 25-gauge needles produced an average cellularity of 1.9216. To the best of our knowledge, only one comparison study on US-guided FNA of STNs for LBC used the pain scale11. In that study using the one sampling technique, the mean pain score in the 23-gauge needle group (1.4 ± 1.1) was found to be lower than that in the 21-gauge needle group (1.8 ± 1.3), although this difference was not statistically significant11. In our study, although the pain score in the 23-gauge needle group (2.1 ± 1.3) was higher than that in the 25-gauge needle group (1.6 ± 1.3), the difference was not statistically significant. The reason for higher pain scores in the 23-gauge needle group may be associated with the smaller nodule sizes in this group than those in the 25-gauge needle group. Thus, we believe that when using the one sampling technique in US-guided FNA of STNs, patient pain does not differ in the use of needles between 21- and 25-gauge.A disadvantage of using larger needles is that they could result in more patient discomfortThe following are the limitations of our study. First, there was a small sample size. Furthermore, the mean nodule size was different between two groups. This may be associated with a sampling bias. Second, 21 FNA cases with purely calcified thyroid nodules, thyroid cysts, or partially cystic thyroid nodules were excluded. Third, we did not investigate all the gauges of needles available (21 to 27-gauge). Fourth, the length of the 25-gauge needle was longer than that of the 23-gauge needle. This difference may have resulted in a bias. Finally, the histopathologic results of the 99 STNs were not investigated, as most of them were not confirmed by surgery.In conclusion, the study results demonstrated that no significant differences were found in the cytological adequacy, cellularity, and pain scores between 23- and 25-gauge needles in US-guided FNA of STNs using LBC. Thus, we believe that both 23- and 25-gauge needles can be used in US-guided FNA of STNs using LBC.


The purpose of this study was to examine the effects of a ballistic or non-ballistic concentric-only bench press (COBP) on subsequent plyometric push-up performance. Fourteen resistance trained men completed two separate one-repetition-maximum (1RM) testing sessions followed by three randomized experimental explosive push-up sessions. These sessions combined a heavy concentric bench press with plyometric push-ups. Using a series of 3 × 10 (condition × time) repeated measures ANOVA, comparisons were made between the effects of ballistic and non-ballistic bench presses on performance of plyometric push-ups to investigate push-up performance variables. Compared with the control condition, both ballistic and non-ballistic bench presses produced lower net impulse and take-off velocity data. No differences were found between ballistic and non-ballistic conditions comparing net impulse and take-off velocity. We conclude that the magnitude of loading used in the current investigation may have caused acute fatigue which led to lower push-up performance characteristics. This information can be used to alter loading protocols when designing complexes for the upper body, combining the bench press and plyometric push-ups. A key performance outcome of a strength and conditioning program is the development of muscular power . Power iPotential reasons for mixed results regarding potentiation may be due to the characteristics of the participants or the design of the potentiation complex itself . SpecifiWhile previous research has examined the differences in potentiation following lower-body exercises performed with ballistic and non-ballistic intent, limited research has examined these differences using upper-body exercises . Given tTo explore the effects of ballistic or non-ballistic COBP on plyometric push-up performance, a repeated-measures design was implemented. Participants reported to the laboratory a total of five times for a measurement of two, one-repetition-maximum sessions (1RM) and three plyometric push-up conditions . The first two sessions were separated by one week to prevent fatigue and the remaining three sessions were randomized in an attempt to prevent any effect of order on performance and were separated by 72–96 h. Participants were asked to refrain from any upper body training at least 72 h before any testing session to prevent any fatigue effects. A series of 3 (condition) × 10 (time) repeated-measures analyses of variance (ANOVA) were used to compare net impulse and take-off velocity between the different testing conditions.Fourteen men  volunteered for this study. Participants were required to have resistance training experience for a minimum of one year, used a program that contained the bench press exercise, and be over the age of 18. Participants were excluded if they reported any orthopedic injuries in the past six months or did not complete the bench press on a frequent, regular basis. All participants read and signed a written informed consent form and all procedures were approved by the university’s Institutional Review Board. The Institutional Review Board approval number for this study is 171027A.The purpose of the bench press 1RM session was to determine the participants’ maximal bench press strength and record the safety bar height for the 1RM concentric-only bench press session. Before conducting the 1RM test, each participant completed a general dynamic warm-up. The warm-up consisted of 25 jumping jacks, 10 forward arm circles, 10 backward arm circles, 10 shoulder taps and 10 push-ups. Following the warm-up, two minutes of passive rest was given before initiating the 1RM test. The 1RM bench press test was implemented by adapting procedures from Suchomel et al. . ParticiFollowing the 1RM bench press session, the researchers determined the height of the safety pins for the next testing session for the 1RM concentric-only bench press. The laboratory assistants lowered the safety pins so that when the barbell was at the lowest position on the safety pin, it simultaneously rested on the participant’s sternum. This height for the safety pins was recorded and used for the remainder of the study.One week following the 1RM bench press session, subjects reported to the laboratory at approximately the same time of day to determine their COBP 1RM from safety pins. The purpose of this session was to determine the weights that the participant would use under the experimental conditions and familiarize them with the concentric-only bench press from pins. The same general dynamic warm-up and 1RM protocol listed previously were used for this session. All repetitions of the COBP were performed with the barbell resting on the safety pins and the participant’s sternum. The participants then performed a COBP with full extension of the arms to complete each repetition. Each participant used progressively heavier loads, increased by a minimum of 2.5 kg for each attempt and a maximum was reached in less than 5 maximal attempts. After completing the 1RM and self-selected rest, participants practiced with each of the experimental conditions . During the ballistic condition, participants were instructed to “attempt to throw the bar but don’t let go” and were allowed 5 practice repetitions with a 20 kg barbell. During the non-ballistic conditions, participants were instructed to “press the bar” and were allowed 5 practice repetitions with a 20 kg barbell. Following the practice repetitions, the participant’s self-selected push-up hand placement on the dual force plates  was determined. The distance from the top and side of the dual force plates to the distal end of the third digit was recorded and used for the remainder of the study. Hand placement and start position are shown in Before starting the control testing session, participants completed the same general dynamic warm-up that was completed during the 1RM testing sessions. After completing the warm-up, participants were given one minute of passive rest before starting the plyometric push-up repetitions. Participants completed 1 explosive push-up with 1 minute of rest in between each repetition for 10 total trials. The participants were given the verbal cues “get set” where they placed their hands on the force plate in the bottom position and then received a countdown “3, 2, 1, up!”. Then, participants performed a plyometric push-up with their hands leaving the platform and then landing back onto the force plate under control. Participants completed a concentric-only plyometric push-up in this same manner for all trials. Consistent hand placement was ensured by using markers on the force plate based on the familiarization session. The two experimental sessions and control sessions were randomized and completed in the same manner for all testing conditions. Following the same warm-up described above, participants completed a COBP potentiation protocol with ballistic or non-ballistic intent following 1 min of passive rest. The COBP potentiation protocol consisted of 5 repetitions at 30%, 3 repetitions at 50%, 3 repetitions at 70%, and 2 repetitions at 90% of the previously tested 1RM COBP. Participants rested for one minute between 30% and 50%, 2 minutes between 50% and 70%, and 3 min between 70% and 90%. Participants were given verbal encouragement on all repetitions based on the script detailed above. After the final COBP repetition, one minute of rest was provided prior to the participant performing 10 plyometric push-ups on the dual force plates in the same manner as the control testing session. All push-up repetitions were performed on dual force plates sampled at 1000 Hz. The data were collected using NetForce software . All data were analyzed using a custom written MATLAB program . p ≤ 0.05 as a standard of statistical significance.Normality of the data was assessed using the Shapiro–Wilk test. Test–retest reliability of net impulse and take-off velocity was assessed using the control condition data with two-way mixed intraclass correlation coefficients. To examine the potential differences between the control, ballistic, and non-ballistic conditions, two sets of 3 (condition) × 10 (time) repeated measures ANOVA were used. If statistical significance was present for the main effect or interaction data, Bonferroni post hoc analyses with 95% confidence intervals (CI) were used. If the assumption of sphericity was violated, Greenhouse–Geisser adjusted values were used. Practical significance at each time interval between conditions was assessed using Cohen’s d effect sizes. Effect size magnitudes of 0–0.19, 0.20–0.59, 0.60–1.19, 1.20–1.99, 2.0–3.99, and ≥4.0 were interpreted as trivial, small, moderate, large, very large, and nearly perfect, respectively . All staNet impulse and take-off velocity data were normally distributed. Test–retest reliability of the net impulse and take-off velocity data ranged from 0.95 to 0.96.p < 0.001, power = 0.99) and take-off velocity . Post hoc analysis indicated that the control condition produced greater time-averaged net impulse characteristics compared to the ballistic  and non-ballistic conditions . In contrast, no statistical differences existed between the ballistic and non-ballistic conditions . Similar to net impulse, post hoc analysis indicated that the control condition produced greater time-averaged take-off velocities compared to both the ballistic  and non-ballistic conditions . No statistical differences existed between the ballistic and non-ballistic conditions for time-averaged take-off velocity .A statistically significant main effect of the conditions existed for both net impulse  and take-off velocity . Post hoc analysis indicated that the condition-averaged net impulse produced at minute 2, post-stimulus, was statistically greater than the magnitude produced at minute 9, post-stimulus . However, no other statistically significant differences existed between time intervals (p > 0.05). In reference to condition-averaged take-off velocity, the magnitude produced at minute 1, post-stimulus, was statistically greater than the magnitude produced at minute 9, post-stimulus . Similarly, the take-off velocity produced at minute 2, post-stimulus, was statistically greater than the magnitude produced at minute 9, post-stimulus, . No other statistically significant differences existed between time intervals (p > 0.05).A statistically significant main effect of time existed for both net impulse  and take-off velocity . The effect size differences between the control condition and the testing condition at each time interval are presented in p = 0.003), but not for the ballistic (p = 0.269) or non-ballistic conditions (p = 0.080). Specifically, net impulse at 2  and 4 minutes , post-stimulus, was statistically greater than the net impulse at 9 minutes, post-stimulus, while no other statistically significant differences existed (p > 0.05) during the control condition. It should be noted that the effect sizes ranged from trivial to moderate compared to the lowest net impulse at 9 min, post-stimulus (d = 0.14–0.98), dependent on the rest interval. Similar to net impulse, post hoc analysis revealed statistically significant differences within the control condition for take-off velocity (p = 0.005), but not for the ballistic (p = 0.342) or non-ballistic conditions (p = 0.178). A specific difference included a greater take-off velocity at 4 minutes, post-stimulus, compared to 9 min, post-stimulus ; however, no other differences between rest intervals existed (p > 0.05). It should be noted that effect sizes compared to the slowest take-off velocity ranged from trivial to moderate (d = 0.18–1.08), dependent on the rest interval.Statistically significant condition x time interaction effects existed for both net impulse (The purpose of this investigation was to compare the acute effect of ballistic and non-ballistic COBP on subsequent plyometric push-up performance. Specifically, the authors endeavored to compare ballistic with non-ballistic COBP on net-impulse and take-off velocity in explosive push-ups. The main outcomes from this study indicated that, compared to a control session, both experimental conditions produced smaller magnitudes of net impulse and take-off velocity, while no differences existed between the ballistic and non-ballistic conditions. Prior research investigating upper-body potentiation complexes has demonstrated improvements in upper-body explosive performance ,9,10,16.In a series of lower-body studies, Suchomel et al. ,7,26, deThis study had several limitations to consider. First, there was a relationship between strength levels and acute performance outcomes after heavy loading . The parThe results of the present investigation indicated that heavy COBP preceding plyometric push-ups did not elicit a performance improvement. Furthermore, performing the bench press in a ballistic or non-ballistic manner did not alter any of the measured performance variables. Although performance outcomes were not obtained from this loading prescription, future studies should examine the effects of a lighter magnitude of loading prior to plyometric push-ups. Finally, coaches should be aware that loading prescription used for lower-body muscle groups may differ from the muscles of the upper-body and may necessitate alterations in a training protocol.